Update of WHO biosafety risk assessment and guidelines for the production and quality control of human
influenza vaccines against avian influenza A(H7N9) virus
As of 10 May 2013
Introduction
This document updates WHO guidance to national regulatory authorities and vaccine manufacturers on the safe production and quality control
of human influenza vaccines produced in response to a pandemic threat1. It details international biosafety expectations for both pilot-scale
and large-scale production, and quality control of vaccines against avian influenza A(H7N9) virus now causing human infections in China. The
updated guidance applies to both candidate vaccine development and to production activities for inactivated and live attenuated influenza
vaccines. The general guidance in this document should be supplemented by specific risk assessments that should be carried out by
laboratories or manufacturers intending to work with H7N9 virus-derived candidate vaccine viruses (CVV) and are to be updated as new
characteristics of the viruses become available.
Development of the document
A small expert group convened by WHO held "virtual" consultations over the period 12 April – 6 May 2013. The group included biosafety
experts, influenza virologists, veterinarians, representatives from laboratories involved in developing the vaccine virus strains and experts from
the animal—human interface field2. The group was asked to address questions about (1) testing of the CVVs being considered for human
1
WHO biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines, TRS No. 941, Annex 5 (2007),
http://www.who.int/biologicals/publications/trs/areas/vaccines/influenza/Annex%205%20human%20pandemic%20influenza.pdf, accessed 03 May 2013
2
The experts are listed in the Acknowledgements section. Each expert had completed a Declaration of Interest form and none had conflicts of interest concerning
development of this guidance.
1
vaccine production; and (2) risk assessment for vaccine production of human vaccines to protect against H7N9 virus. The conclusions reached
by the expert group form the basis of this updated guidance from WHO.
Background information
On 31 March 2013, WHO was informed by China of cases of human infections with avian influenza A(H7N9) virus. Since then, 128 human cases
have been confirmed in China3. Related viruses have been identified in avian species, including pigeons, chickens and ducks.
The updated recommendations are predicated on the demonstration that the avian influenza A(H7N9) viruses are of low pathogenicity in
chickens. The latter conclusion is based on the following evidence: (i) notifications that the virus currently fits the OIE definition of low
pathogenicity avian influenza for poultry according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals4; (ii) sequence
analysis of the H7N9 viruses demonstrated that the haemagglutinin (HA) gene is consistent with phenotype of low pathogenicity in poultry 5
(no accumulation of basic or other amino acids at the HA cleavage site); (iii) H7N9 viruses have been detected in live-bird markets and samples
from poultry showing no overt disease signs.
At the time of this writing, preliminary results from multiple laboratories using the standard pathogenicity test (outlined in Appendix 1)
indicate that H7N9 wild-type virus replicates efficiently in the upper and lower respiratory tract of infected ferrets, and may cause signs of
disease, including weight loss, fever and transient lethargy, but does not cause severe or fatal disease.
3
For up-to-date information, see: http://www.who.int/influenza/human_animal_interface/influenza_h7n9/en/index.html, accessed 03 May 2013
‘On 4 April 2013, the Chinese Veterinary Authorities notified the occurrence of infection of pigeons and chickens with low pathogenic avian influenza virus H7N9 to the
OIE.’, http://www.oie.int/for-the-media/press-releases/detail/article/questions-and-answers-on-influenza-ah7n9/, accessed 03 May 2013; and subsequent follow-up
4
reports http://www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapFullEventReport&reportid=13391, accessed 7 may 2103
5
http://www.oie.int/for-the-media/press-releases/detail/article/questions-and-answers-on-influenza-ah7n9/, accessed 03 May 2013; and WHO RISK ASSESSMENT Human
infections with influenza A(H7N9) virus) http://www.who.int/entity/influenza/human_animal_interface/influenza_h7n9/RiskAssessment_H7N9_13Apr13.pdf accessed 06
May 2013
2
Limited information is available on the pathogenicity of H7N9 viruses in humans. A substantial number of human cases have been severely ill,
and many deaths have occurred, while a few milder cases have also been identified.
Testing of candidate vaccine viruses (CVV) considered for vaccine production
Taking into account the currently limited information about the pathogenicity of H7N9 viruses as well as the experience gained over decades
with reassortant CVV incorporating HA and neuraminidase (NA) genes derived from both highly pathogenic avian influenza (HPAI) and low
pathogenic avian influenza (LPAI) viruses, the experts consulted by WHO concluded that the following tests should be conducted on
reassortant CVV:




Sequence analysis of the HA gene to confirm the presence of a cleavage site consistent with low pathogenic influenza viruses ;
An assay to detect the ability of the virus to cause chicken embryo death;
Pathogenicity test in ferrets; and
An assay to determine the genetic stability of the HA gene of the virus upon multiple passaging.
A CVV is considered attenuated if the above tests show that (i) the sequence of the HA cleavage site is consistent with phenotype of low
pathogenicity; (ii) the CVV does not cause embryo lethality; (iii) the pathogenicity of the CVV in ferrets is lower than that of the wild-type H7N9
virus from which it is derived6; and (iv) the CVV has been shown to maintain an HA cleavage site consistent with low pathogenic phenotype
upon multiple passage in embryonated chicken eggs.
6
A genetically related wild-type virus may be used as comparator if its use is appropriately justified.
3
Such attenuated CVV should be handled in accordance with the recommendations described for ‘CVV’ in this document (see section Risk
assessment for work with CVV derived from H7N9 viruses) and are distinguished from ‘potential CVVs’7, for which the results of these tests
have not been obtained or are unavailable.
Notes:
The expert group recommended that these tests be carried out on newly developed reassortant viruses, generated either by conventional
reassortment or by reverse genetics, including, in the latter case, those viruses derived from synthetic nucleic acid. However, reassortant
viruses that have HA and NA genes with sequences that are identical or nearly identical to those of a reassortant virus that has already been
assessed using these tests may be exempted from safety testing. A review of safety data obtained from the first few reassortant CVV will be
performed by the expert group to determine whether or not the suggested testing regimen should be changed.
The schedule of testing is based on the finding that the parental wild-type H7N9 viruses are of low pathogenic phenotype in chickens, as
assessed according to the Manual for Diagnostic Tests and Vaccines for Terrestrial Animals 2012 8. Should highly pathogenic H7N9 viruses
emerge and be used for the derivation of reassortant CVV , the testing regimen described here would need to be re-evaluated; it is expected
that in this case, testing as prescribed in TRS No 941, Annex 5 (2007) (sections 2.6.1 and 2.6.2) for reassortant viruses derived from highly
pathogenic avian viruses of subtypes H5 or H7 would be appropriate, including pathogenicity testing in chickens1.
In addition to the determination of the amino acid sequence of the HA cleavage site by sequencing of the HA gene, particular attention should
be given to amino acid substitutions in genes derived from the wild-type H7N9 parent virus that indicate potential adaptation to mammalian
hosts. Sequence information currently available for H7N9 viruses is indicative of some level of mammalian adaptation of these viruses; if any
further adaptive changes are identified in CVV, a review of all safety data should be conducted.
7
‘ Vaccine response to the avian influenza A(H7N9) outbreak – step 1: development and distribution of candidate vaccine viruses’ (2 May 2013),
http://www.who.int/influenza/vaccines/virus/CandidateVaccineVirusesH7N9_02May13.pdf, accessed 03 May 2013
8
Manual for Diagnostic Tests and Vaccines for Terrestrial Animals 2012; OIE. http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/,
accessed 03 May 2013
4
It is recognized that the test for genetic stability takes a considerable amount of time and could delay the distribution of newly developed CVV.
Work to date with CVV derived from highly pathogenic avian A(H5N1) viruses with modified HA genes (removal of polybasic cleavage site) has
shown that these have, in all cases where this was assessed, retained their attenuated phenotype. It is therefore recommended that this test
be carried out in parallel with other safety testing and in parallel with distribution of CVV. If loss of the attenuated phenotype, i.e. insertion of
additional amino acids at the HA cleavage site, were identified for any newly developed H7N9 CVV during testing for genetic stability, an
urgent signal would be sent to all recipients of the respective virus, through existing channels of communication used by WHO, WHO
Collaborating Centres and WHO Essential Regulatory Laboratories.
Virus transmissibility is an additional aspect in the risk assessment of work conducted with influenza viruses. However, standard tests for
evaluating this characteristic have not been validated and these tests have not been prescribed in previous guidance1,9.The expert group
concluded that, while it would be desirable to obtain data on the transmissibility of H7N9 wild-type viruses and CVV, formal testing of
transmissibility would not be required for CVV derived from H7N9 viruses.
Risk assessment for work with candidate vaccine viruses (CVV) derived from avian A(H7N9) viruses
The expert group recommends the following assignments of containment levels for work with H7N9 viruses:

For small-scale laboratory work:
- containment requirements for work with wild-type H7N9 virus should be BSL3, as specified elsewhere10
9
WHO biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines: Update, 23 July 2009,
http://www.who.int/biologicals/areas/vaccines/influenza/CP116_2009-2107_Biosafety_pandemicA_H1N1_flu_vaccines-Addendum-DRAFTFINAL.pdf, accessed 03 May
2013
10
Biosafety guidelines for handling highly pathogenic avian influenza viruses in veterinary diagnostic laboratories, in the Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals 2012, Chapter 2.3.4. adopted May 2012, Appendix 2.3.4.1,http://www.oie.int/en/international-standard-setting/terrestrial-manual/access-online/
5
- containment requirements for potential CVV, intended for use in the production of inactivated or live attenuated vaccines, should be
as for wild-type viruses

For large-scale work (pilot-scale vaccine production and commercial-scale vaccine production):
- containment level for work with wild-type H7N9 virus, or potential CVV intended for use in the production of inactivated or live
attenuated influenza vaccines, should be BSL-3 enhanced1,10
- containment level for work with characterized CVV (i.e. the attenuation of which has been demonstrated), intended for use in the
production of either inactivated or live attenuated influenza vaccines, should be BSL-2 enhanced1
The risk assessment underlying the present update took account of a number of characteristics of H7N9 virus that were known or imputed
from preliminary data at the time of writing.
The general guidance in this document should be supplemented by specific risk assessments that should be carried out by laboratories or
manufacturers intending to work with H7N9 virus-derived CVV and are to be updated as new characteristics of the agents become available.
The elements of risk assessments to be incorporated can be found in the Laboratory Biosafety Manual, Third Edition,11 and in the Laboratory
Biorisk Management Standard12. Risk assessments need to take into account, in addition to factors specific for the type of work and facility, the
following non-exclusive list of virus characteristics and parameters in relation to H7N9 virus:
11
‘WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus’
http://www.who.int/influenza/resources/documents/guidelines_handling_specimens/en/, accessed 02 May 2013
12
COMITÉ EUROPÉEN DE NORMALISATION Laboratory biorisk management - Guidelines for the implementation of CWA 15793:2008
6



Receptor binding properties of parental H7N9 virus: currently available sequence information suggests that these viruses can bind to
mammalian-type receptors (α 2,6-linked sialic acid residues); this suggests that infectivity for and transmissibility to and between
humans and other mammals may be higher than for avian influenza viruses considered in the past, such as avian H5N1 viruses.
Amino acid residues at various positions in a number of genes of H7N9 virus suggest adaptation to mammalian hosts; if these genes are
present in a reassortant CVV, their potential impact on infectivity and transmissibility should be considered.
Passaging of viruses in various substrates can lead to the acquisition of sequence changes; while the HA gene of H7N9 virus is of the low
pathogenicity type (no accumulation of basic or other amino acids at the HA cleavage site), it cannot be assumed that this genotype is
stable.
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Appendix 1 Safety Testing of Avian Influenza A(H7N9) Vaccine Viruses in Ferrets
Test virus
The 50% embryo or tissue culture infectious dose (e.g. EID50, TCID50) or plaque-forming units (PFU) of the reassortant candidate vaccine virus
(CVV) and parental virus stock, or genetically similar wild-type virus will be determined. The infectivity titres of viruses should be sufficiently
high such that these viruses can be compared using equivalent high doses in ferrets (107 to 106 EID50, TCID50 or PFU) and diluted not less than
1:10. Where possible, the pathogenic properties of the donor PR8 should be characterized thoroughly in each laboratory.
Laboratory facility
Animal studies with the vaccine strain and the parental wild-type strain should be conducted in animal biosafety level (BSL)-3 containment
facilities with enhanced practices1. An appropriate occupational health policy should be in place2.
Experimental procedure
Outbred ferrets 4-12 months of age that are serologically negative for currently circulating influenza A and B viruses and the test virus strain
are anesthetized by either intramuscular administration of a mixture of sedatives (e.g. ketamine (25 mg/kg) and xylazine (2 mg/kg)) and
atropine (0.05 mg/kg) or by suitable inhalant anesthetics. A standard virus dose of 10 7 to106 EID50 (or TCID50 or PFU) in 0.5 to 1 ml of
phosphate-buffered saline is used to inoculate animals. The dose should be the same as that used for pathogenesis studies with the wild-type
parental virus. The virus is slowly administered into the nares of the sedated animals, reducing the risk of virus being swallowed or expelled. A
group of four to six ferrets should be inoculated. One group of two to three animals should be euthanized on day 3 or 4 after inoculation and
samples should be collected for estimation of virus replication from the following organs in order: spleen, intestine, lungs (samples from each
lobe and pooled), brain (anterior and posterior sections sampled and pooled), olfactory bulb of the brain, and nasal turbinates. If gross
pathology demonstrates lung lesions similar to those observed in wild-type viruses, additional lung samples may be collected and processed
with haematoxylin and eosin (H&E) staining for histopathological evaluation. The remaining animals are observed for clinical signs, which may
include weight loss, lethargy [based on a previously published index3], respiratory and neurologic signs, and increased body temperature.
8
Collection of nasal washes from animals anesthetized as indicated above should be performed to determine the level of virus replication in the
upper airways on alternate days after inoculation for up to nine days. At the termination of the experiment on day 14 after inoculation, a
necropsy should be performed on at least two animals and organs collected. If signs of substantial gross pathology are observed (e.g. lung
lesions), the organ samples should be processed as described above for histopathology.
Expected outcome
Clinical signs of disease such as lethargy and/or weight loss should be attenuated in the vaccine virus strain compared with the parental virus
strains, assuming that the parental H7N9 donor virus also consistently causes these signs. Viral titres of the vaccine strain in respiratory
samples should be no greater than either parental strain; a substantial decrease in lung virus replication is anticipated. Gross lung lesions seen
at necropsy should be minimal. Replication of the candidate vaccine virus should be restricted to the respiratory tract. Virus isolation from the
brain is not expected. However, detection of virus in the brain has been reported for seasonal H3N2 viruses4. Therefore, should virus be
detected in the anterior or posterior regions of the brain (excluding the olfactory bulb) the significance of such finding may be confirmed by
performing histopathological analysis of brain on day 14 after inoculation. . Neurological lesions detected in H&E-stained sections should
confirm virus replication in the brain and observation of neurological signs. Presence of neurological signs and histopathology would indicate a
lack of suitable attenuation of the vaccine candidate virus.
1.
‘Biosafety guidelines for handling highly pathogenic avian influenza viruses in veterinary diagnostic laboratories’ in the Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals 2012, Chapter 2.3.4. adopted May 2012, Appendix 2.3.4.1,
http://www.oie.int/en/international-standard-setting/terrestrial-manual/access-online/, accessed 02 May 2013
2.
‘WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus’
http://www.who.int/influenza/resources/documents/guidelines_handling_specimens/en/, accessed 02 May 2013
9
3.
Reuman PD, Keely S, and Schiff GM. (1989). Assessment of signs of influenza illness in the ferret model. Laboratory Animal Science
42:222-232.
4.
Zitzow LA, et al., (2002) Pathogenesis of influenza A (H5N1) viruses in ferrets. Journal of Virology 76:4420-4429.
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Table 1: Summary of results in ferrets infected intranasally with XXXX influenza A(H7N9) candidate vaccine virus
No. animals with clinical signs to
Respiratory tract viral titers
(log10EID50/ml or g)c
day 14 p.i.
Other (e.g.
fever)
Dose
(EID50)b
No.
Animals
Lethargy
Respiratory
Mean max. %
weight loss
Weight
loss
Virus
Mean peak
nasal wash or
nasal
turbinate
Lung
lesions
Lung
lesions
(day 3/4)d,e
(day 14)d,e
Lung
Detection of
virus in other
organf
XXXX
A/Shanghai/2/2013a
wt A/Anhui/1/2013
hg or ca parental donor (e.g.
A/PR/8/34)
a
b
c
A/Anhui/1/2013 is antigenically and genetically similar to the parental wild-type donor virus A/Shanghai/2/2013.
Indicate whether dose is expressed as EID50, TCID50 or pfu.
Indicate whether respiratory viral titers are expressed as EID50, TCID50 or pfu per ml or g. Give lower limit of detection.
11
d
Score gross pathologic lung lesions as -, + (≤20%), ++ (>20 and < 70%), +++ (>70%)
e
Indicate outcome of any histopathology evaluation
f
Indicate organ or not detected
p.i., post inoculation
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Acknowledgments
The ad hoc expert group on WHO biosafety guidelines on production and quality control of H7N9 vaccines is composed of the following
experts (alphabetically):
Dr Othmar G Engelhardt, Division of Virology, National Institute for Biological Standards and Control, Medicines and Healthcare
Products Regulatory Agency, United Kingdom; Dr Mary Louise Graham, Office of Biosafety and Biocontainment Operations, Pathogen
Regulation Directorate, Public Health Agency of Canada, Canada; Dr Gary Grohmann, Office of Laboratories and Scientific Services,
Monitoring and Compliance Group, Therapeutic Goods Administration, Australia; Dr Shigeyuki Itamura, Laboratory for Quality Control
of Influenza Vaccine, Center for Influenza Virus Research, National Institute of Infectious Diseases, Japan; Dr Jacqueline M. Katz,
Epidemiology and Prevention Branch, Influenza Division, Centers for Disease Control and Prevention, United States of America; Dr Xin
Li, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention P. R. China; Dr Kathrin
Summermatter, Institute for Virology and Immunoprophylaxis, Switzerland; Dr Richard Webby, Department of Infectious Diseases, St.
Jude Children’s Research Hospital, United States of America; Dr Jerry Weir, Division of Viral Products, Center for Biologics Evaluation
and Research, Food and Drug Administration, United States of America; Dr Gert Zimmer, Institute for Virology and Immunoprophylaxis,
Switzerland.
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